The cellular and molecular mechanisms underlying age-dependent neurodegeneration seen in metazoans from worms to humans are poorly understood. Neurodegeneration is largely genetic in origin and often results from a single gene defect. (reviewed in FORTINI and BONINI 2000; HEINTZ and ZOGHBI 2000; FEANY 2000; FORMAN et al. 2000; GOEDERT 2001; SIPIONE and CATTANEO 2001; MACCIONI et al. 2001). Human neurodegenerative illnesses, such as Amyotropic Lateral Sclerosis (ALS), Huntington disease, Parkinson disease, and Alzheimer disease are characterized by progressive behavioral deficits, premature death and, in some cases, profound cognitive impairment. Onset of the symptoms of these diseases correlates with the appearance of neuropathology. Mutations that underlie some forms of these diseases are known, but a general understanding of the molecular mechanisms required for maintaining neuronal viability are not understood.
A great demand exists for therapeutic interventions for neurodegenerative diseases, particularly for administration to elderly subjects. Notwithstanding the great demand, few if any drugs are available to reduce age-related neurodegeneration. At the heart of this lack of therapeutic agents is the lack of adequate screening assays for novel therapeutic interventions. Typically, neuroblast-derived cell lines in culture are exposed to putative agents and agents that extend the life in culture are selected for further investigation. What is missing is an efficient in vivo technology for use as a primary screen or for confirming effectiveness of putative neuroprotective agents.
Human neurodegenerative conditions can be modeled in Drosophila. In some cases, human proteins, such as alpha-synuclein and tau, are expressed in Drosophila and cause neurodegenerative syndromes having phenotypic properties similar to those of Parkinson and Alzheimer diseases, respectively. Likewise, flies expressing human Huntingtin containing expanded triplet repeats develop neuropathological defects reminiscent of human Huntington disease. Using this model system, various suppressor mutations have been isolated. It has further been determined that onset of neurodegeneration in flies can be suppressed by overexpressing human hsp70. Accordingly, it appears that Drosophila can be instrumental in uncovering key mechanisms of general significance in the field of neurodegenerative disorders.
Additionally, Drosophila have been screened for single gene mutations that cause neurodegeneration. Mutants such as drop dead, swiss cheese, eggroll, spongecake, and bubblegum have moderate to markedly reduced lifespans and associated neuropathology including vacuolization and accumulation of multi-lamellar cell bodies. These neuropathologies are similar to those seen in patients having Tay-Sachs and Creutzfeldt-Jakob diseases.
Still, the number of neurodegeneration mutants is quite small and additional neurodegeneration mutants are of interest. Such mutants not only help in deciphering basic neurodegeneration biology, but can also serve as convenient and inexpensive models both for genetic therapies and for screening putative neuroprotective agents. It is, of course, difficult to ascertain which strains of Drosophila exhibit neurodegeneration. It is impractical to screen all strains and mutants for neurodegeneration and the art lacks a principled basis upon which one would select candidates a priori. Prior efforts have examined flies on the basis of a defect in phototaxis or reduced life span. (HEISENBERG and BOHL, 1979; HEISENBERG 1979; COOMBE and HEISENBERG 1986; BUCHANAN and BENZER 1993; MIN and BENZER 1997; KRETZSCHMAR et al. 1997). For example, Min and Benzer, 1997 examined five thousand mutagenized lines and isolated sixty mutant lines having reduced lifespan. Of the sixty mutants, two (0.4% of the mutagenized lines examined) exhibited neurodegeneration. Screening for mutant lines having reduced life span presents at least two inherent disadvantages, namely the substantial time required to ascertain a shortened life span and the low incidence in such mutants of neurodegeneration. A more targeted and efficient approach to screening for neurodegeneration mutants in Drosophila is desired.
Previous studies have established a connection between neuronal dysfunction and neurodegeneration in some species. Some neurodegenerative mutants affect genes that encode ion channels and neurotransmitter receptors. Weaver (wv), lurcher (Lc), and tottering (tg) were identified in mice on the basis of locomotor behavior defects and contain mutations in genes that encode ion channels and neurotransmitter receptors. Studies of these mutants have demonstrated an important connection between aberrant neuronal signaling properties and neurodegeneration (MURTOMAKI et al. 1995; NORMAN et al. 1995; FLETCHER et al. 1996; ZUO et al. 1997). The connection is also established in the worm sensory system (HALL et al. 1997). An indirect connection between neurodegeneration and ion channels is seen in Drosophila dADAR mutants which exhibit extensive neurodegeneration arising from lack of an enzyme essential for adenosine to inosine type editing of pre-mRNAs that encode several Drosophila ion channels. Notably, dADAR null mutants undergo extensive neurodegeneration (PALLADINO et al. 2000a).
This application also describes various mutations in the alpha subunit of Na+/K+ ATPase pumps (sodium pumps) that asymmetrically distribute Na+ and K+ ions to form ion gradients across the plasma membrane of cells. These ion gradients determine the membrane resting potential and excitability of cells and drive many important secondary processes. Without such ion gradients, many essential functions, including electrical signaling in the nervous system, are not possible. Many sodium pump isozymes exist, are highly conserved evolutionarily, and are widely expressed in animal tissues. In neurons, sodium pumps generate and maintain the membrane potential after extensive Na+ influx enabling continued generation of action potentials. Not surprisingly, sodium pumps are extensively regulated in vivo (reviewed in THERIN and BOLSTEIN, 2000).
Sodium pumps have at least two essential subunits, alpha and beta. The alpha subunit of the Drosophila Na+/K+ ATPase (ATPalpha) is a large protein (>110 kDa) with multiple transmembrane domains and an ATP-dependent catalytic activity. A version of Drosophila ATPalpha is available in Genbank at Accession No. XP-081160, presented herein as SEQ ID NO:1. Mutations and reversions described herein are defined relative to the ATPalpha amino acid sequence disclosed in Accession No. XP-081160 and are not separately presented. It is understood that the skilled artisan can readily understand the complete sequences of mutants and reversions from the information presented in the specification. The beta subunit has a single transmembrane domain and may be involved in pump maturation, membrane localization and functional properties of Na+/K+ ATPases.
Studies characterizing the functions and importance of Na+/K+ ATPase proteins in vivo in other animals are limited but suggest that normal neural development and maintenance requires proper Na+/K+ ATPase function. One study of Na+/K+ ATPase loss-of-function eat-6 mutations in the nematode established a link between pharyngeal function and sodium pump activity. Null mutations of the mouse Na+/K+ ATPase beta2 subunit cause neural cell degeneration, apoptotic photoreceptor cell death, and death late in development.
The importance of Na+/K+ ATPase function has also been suggested by widespread expression in metazoan tissues, striking evolutionary conservation, and involvement in many essential processes including nutrient absorption, nephritic function and signaling in the nervous system. Many studies suggest a pathophysiological connection between the biochemical function of these important proteins and human neural diseases including bipolar disorder, seizures and neurodegenerative conditions, namely spongiform encephalopathies, with manifestations similar to those caused by prion diseases, namely Kuru, Crutzfeld-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome (reviewed by MOBASHERI et al, 2000).
Na+/K+ ATPase function is also implicated in cardiac hypertrophy, hypertension, renal dysfunction, bipolar mood disorder, and spongiform encephalopathies. Somewhat surprisingly, no direct mutation of Na+/K+ ATPase alpha has been identified as the cause of neural disease.
Among conditional paralytic mutants, mutations are known to cause neuronal dysfunction by disrupting polynucleotides that encode electrical signaling proteins. One bang-sensitive paralytic mutation and several lethal p element insertions have been mapped to ATPalpha (SCHUBIGER, 1994), (FENG, 1997). Additionally, transgenic ATPalpha having specific modifications at a phosphorylation site required during ATP hydrolysis causes bang-sensitive paralysis and, in some cases, death (SUN, 2001).
No association between neurodegeneration and mutations in Na+/K+ ATPase alpha has been reported, although loss of Na+/K+ ATPase function can cause neuropathological effects (reviewed in Beal, 1993; Lees, 1993). These neuropathological effects are seen after administering Na+/K+ ATPase inhibitors or in the presence of mutations that affect Na+/K+ ATPase beta subunits. In addition, inherited defects associated with reduced Na+/K+ ATPase activity have been linked to neonatal status convulsivus, spongiform encephalopathy (RENKAWEK et al, 1992).